High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCßII, and SPAK/OSR1.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

Treatment with the KCa3.1 inhibitor TRAM-34 during diabetic ketoacidosis reduces inflammatory changes in the brain.

BACKGROUND: Diabetic ketoacidosis (DKA) causes brain injuries in children ranging from subtle to life-threatening. Previous studies suggest that DKA-related brain injury may involve both stimulation of Na-K-Cl cotransport and microglial activation. Other studies implicate the Na-K-Cl cotransporter and the Ca-activated K channel KCa3.1 in activation of microglia and ischemia-induced brain edema. In this study, we determined whether inhibiting cerebral Na-K-Cl cotransport or KCa3.1 could reduce microglial activation and decrease DKA-related inflammatory changes in the brain. METHODS: Using immunohistochemistry, we investigated cellular alterations in brain specimens from juvenile rats with DKA before, during and after insulin and saline treatment. We compared findings in rats treated with and without bumetanide (an inhibitor of Na-K-Cl cotransport) or the KCa3.1 inhibitor TRAM-34. RESULTS: Glial fibrillary acidic protein (GFAP) staining intensity was increased in the hippocampus during DKA, suggesting reactive astrogliosis. OX42 staining intensity was increased during DKA in the hippocampus, cortex and striatum, indicating microglial activation. Treatment with TRAM-34 decreased both OX42 and GFAP intensity suggesting a decreased inflammatory response to DKA. Treatment with bumetanide did not significantly alter OX42 or GFAP intensity. CONCLUSIONS: Inhibiting KCa3.1 activity with TRAM-34 during DKA treatment decreases microglial activation and reduces reactive astrogliosis, suggesting a decreased inflammatory response.

Engaging neuroscience to advance translational research in brain barrier biology.

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and--when barrier integrity is impaired--in the pathology of many common CNS disorders such as Alzheimer's disease, Parkinson's disease and stroke.

Diabetic ketoacidosis in juvenile rats is associated with reactive gliosis and activation of microglia in the hippocampus.

BACKGROUND: Type 1 diabetes may be associated with structural and functional alterations in the brain. The role of diabetic ketoacidosis (DKA) in causing these alterations has not been well explored. METHODS: We used immunohistochemical staining to investigate cellular alterations in brain specimens from juvenile rats with DKA before, during, and after treatment with insulin and saline, and compared these to samples from diabetic rats and normal controls. RESULTS: Glial fibrillary acidic protein (GFAP) staining intensity was increased in the hippocampus during DKA and increased further during insulin/saline treatment. Twenty-four and 72 h after treatment, hippocampal GFAP intensity declined but remained above control levels. There were no significant changes in GFAP intensity in the cortex or striatum. OX42 staining intensity was increased during untreated DKA and increased further during insulin/saline treatment in the hippocampus and cortex. NeuN staining intensity was decreased after DKA treatment in the striatum but not in other regions. CONCLUSIONS: DKA causes inflammatory changes in the brain including reactive gliosis and activation of microglia. These findings are present during untreated DKA, but intensify during insulin/saline treatment. The hippocampus was disproportionately affected, consistent with previous studies showing deficits in hippocampal functions in rats after DKA recovery and decreased memory capacity in children with a history of DKA.

Blood-brain barrier KCa3.1 channels: evidence for a role in brain Na uptake and edema in ischemic stroke.

BACKGROUND AND PURPOSE: KCa3.1, a calcium-activated potassium channel, regulates ion and fluid secretion in the lung and gastrointestinal tract. It is also expressed on vascular endothelium where it participates in blood pressure regulation. However, the expression and physiological role of KCa3.1 in blood-brain barrier (BBB) endothelium has not been investigated. BBB endothelial cells transport Na(+) and Cl(-) from the blood into the brain transcellularly through the co-operation of multiple cotransporters, exchangers, pumps, and channels. In the early stages of cerebral ischemia, when the BBB is intact, edema formation occurs by processes involving increased BBB transcellular Na(+) transport. This study evaluated whether KCa3.1 is expressed on and participates in BBB ion transport. METHODS: The expression of KCa3.1 on cultured cerebral microvascular endothelial cells, isolated microvessels, and brain sections was evaluated by Western blot and immunohistochemistry. Activity of KCa3.1 on cerebral microvascular endothelial cells was examined by K(+) flux assays and patch-clamp. Magnetic resonance spectroscopy and MRI were used to measure brain Na(+) uptake and edema formation in rats with focal ischemic stroke after TRAM-34 treatment. RESULTS: KCa3.1 current and channel protein were identified on bovine cerebral microvascular endothelial cells and freshly isolated rat microvessels. In situ KCa3.1 expression on BBB endothelium was confirmed in rat and human brain sections. TRAM-34 treatment significantly reduced Na(+) uptake, and cytotoxic edema in the ischemic brain. CONCLUSIONS: BBB endothelial cells exhibit KCa3.1 protein and activity and pharmacological blockade of KCa3.1 seems to provide an effective therapeutic approach for reducing cerebral edema formation in the first 3 hours of ischemic stroke.

Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase.

Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl, and water into the brain across an intact blood-brain barrier (BBB), together with swelling of astrocytes as they take up the ions and water crossing the BBB. Our previous studies provide evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia, and AVP, and inhibition of NKCC and NHE activities by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. We examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundance. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemic factors. We found that hypoxia, aglycemia, and AVP increase CMEC NHE activity through 5 h and that NHE1, but not NHE2, abundance is increased by 1- to 5-h exposures to these factors. Furthermore, we found that these factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.

Brain cell swelling during hypocapnia increases with hyperglycemia or ketosis.

BACKGROUND: Severe hypocapnia reduces cerebral blood flow (CBF) and is known to be a risk factor for diabetic ketoacidosis (DKA)-related cerebral edema and cerebral injury in children. Reductions in CBF resulting from hypocapnia alone, however, would not be expected to cause substantial cerebral injury. We hypothesized that either hyperglycemia or ketosis might alter the effects of hypocapnia on CBF and/or cerebral edema associated with CBF reduction. METHODS: We induced hypocapnia (pCO2 20 ± 3 mmHg) via mechanical ventilation in three groups of juvenile rats: 25 controls, 22 hyperglycemic rats (serum glucose 451 ± 78 mg/dL), and 15 ketotic rats (ß-hydroxy butyrate 3.0 ± 1.0 mmol/L). We used magnetic resonance imaging to measure CBF and apparent diffusion coefficient (ADC) values in these groups and in 17 ventilated rats with normal pCO2 (40 ± 3 mmHg). In a subset (n = 35), after 2 h of hypocapnia, pCO2 levels were normalized (40 ± 3 mmHg) and ADC and CBF measurements were repeated. RESULTS: Declines in CBF with hypocapnia occurred in all groups. Normalization of pCO2 after hypocapnia resulted in hyperemia in the striatum. These effects were not substantially altered by hyperglycemia or ketosis. Declines in ADC (suggesting brain cell swelling) during hypocapnia, however, were greater during both hyperglycemia and ketosis. CONCLUSIONS: We conclude that brain cell swelling associated with hypocapnia is increased by both hyperglycemia and ketosis, suggesting that these metabolic conditions may make the brain more vulnerable to injury during hypocapnia.

Ischemia-induced stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransport involves p38 and JNK MAP kinases.

Previous studies have provided evidence that, in the early hours of ischemic stroke, a luminal membrane blood-brain barrier (BBB) Na-K-Cl cotransporter (NKCC) participates in ischemia-induced cerebral edema formation. Inhibition of BBB NKCC activity by intravenous bumetanide significantly reduces edema and infarct in the rat permanent middle cerebral artery occlusion model of ischemic stroke. We demonstrated previously that the BBB cotransporter is stimulated by hypoxia, aglycemia, and AVP, factors present during cerebral ischemia. However, the underlying mechanisms have not been known. Ischemic conditions have been shown to activate p38 and JNK MAP kinases (MAPKs) in brain, and the p38 and JNK inhibitors SB-239063 and SP-600125, respectively, have been found to reduce brain damage following middle cerebral artery occlusion and subarachnoid hemorrhage, respectively. The present study was conducted to determine whether one or both of these MAPKs participates in ischemic factor stimulation of BBB NKCC activity. Cultured cerebral microvascular endothelial cell NKCC activity was evaluated as bumetanide-sensitive (86)Rb influx. Activities of p38 and JNK were assessed by Western blot and immunofluorescence methods using antibodies that detect total vs. phosphorylated (activated) p38 or JNK. We report that p38 and JNK are present in cultured cerebral microvascular endothelial cells and in BBB endothelial cells in situ and that hypoxia (7% O(2) and 2% O(2)), aglycemia, AVP, and O(2)-glucose deprivation (5- to 120-min exposures) all rapidly activate p38 and JNK in the cells. We also provide evidence that SB-239063 and SP-600125 reduce or abolish ischemic factor stimulation of BBB NKCC activity. These findings support the hypothesis that ischemic factor stimulation of the BBB NKCC involves activation of p38 and JNK MAPKs.

Ischemia-induced stimulation of Na-K-Cl cotransport in cerebral microvascular endothelial cells involves AMP kinase.

Increased blood-brain barrier (BBB) Na-K-Cl cotransporter activity appears to contribute to cerebral edema formation during ischemic stroke. We have shown previously that inhibition of BBB Na-K-Cl cotransporter activity reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. We have also shown that the BBB cotransporter is stimulated by the ischemic factors hypoxia, aglycemia, and arginine vasopressin (AVP), although the mechanisms responsible are not well understood. AMP-activated protein kinase (AMPK), a key mediator of cell responses to stress, can be activated by a variety of stresses, including ischemia, hypoxia, and aglycemia. Previous studies have shown that the AMPK inhibitor Compound C significantly reduces infarct in mouse MCAO. The present study was conducted to evaluate the possibility that AMPK participates in ischemic factor-induced stimulation of the BBB Na-K-Cl cotransporter. Cerebral microvascular endothelial cells (CMEC) were assessed for Na-K-Cl cotransporter activity as bumetanide-sensitive (86)Rb influx. AMPK activity was assessed by Western blot analysis and immunofluorescence methods using antibodies that detect total versus phosphorylated (activated) AMPK. We found that hypoxia (7% and 2% O(2)), aglycemia, AVP, and oxygen-glucose deprivation (5- to 120-min exposures) increase activation of AMPK. We also found that Compound C inhibition of AMPK reduces hypoxia-, aglycemia-, and AVP-induced stimulation of CMEC Na-K-Cl cotransporter activity. Confocal immunofluorescence of perfusion-fixed rat brain slices revealed the presence of AMPK, both total and phosphorylated kinase, in BBB in situ of both control and ischemic brain. These findings suggest that ischemic factor stimulation of the BBB Na-K-Cl cotransporter involves activation of AMPK.

Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance.

In the early hours of ischemic stroke, cerebral edema forms as Na, Cl, and water are secreted across the blood-brain barrier (BBB) and astrocytes swell. We have shown previously that ischemic factors, including hypoxia, aglycemia, and arginine vasopressin (AVP), stimulate BBB Na-K-Cl cotransporter (NKCC) and Na/H exchanger (NHE) activities and that inhibiting NKCC and/or NHE by intravenous bumetanide and/or HOE-642 reduces edema and infarct in a rat model of ischemic stroke. Estradiol also reduces edema and infarct in this model and abolishes ischemic factor stimulation of BBB NKCC and NHE. There is evidence that NKCC and NHE also participate in ischemia-induced swelling of astrocytes. However, little is known about estradiol effects on astrocyte cell volume. In this study, we evaluated the effects of AVP (100 nM), hypoxia (7.5% O(2)), aglycemia, hypoxia (2%)/aglycemia [oxygen glucose deprivation (OGD)], and estradiol (1-100 nM) on astrocyte cell volume using 3-O-methyl-d-[(3)H]glucose equilibration methods. We found that AVP, hypoxia, aglycemia, and OGD (30 min to 5 h) each significantly increased astrocyte cell volume, and that estradiol (30-180 min) abolished swelling induced by AVP or hypoxia, but not by aglycemia or OGD. Bumetanide and/or HOE-642 also abolished swelling induced by AVP but not aglycemia. Abundance of aquaporin-4, known to participate in ischemia-induced astrocyte swelling, was significantly reduced following 7-day but not 2- or 3-h estradiol exposures. Our findings suggest that hypoxia, aglycemia, and AVP each contribute to ischemia-induced astrocyte swelling, and that the edema-attenuating effects of estradiol include reduction of hypoxia- and AVP-induced astrocyte swelling and also reduction of aquaporin-4 abundance.

Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin.

Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke by increased transport of Na from blood into brain across an intact BBB. We previously showed that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently, we found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchange protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB Na/H exchange activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion-fixed rat brain revealed that Na/H exchanger isoforms 1 and 2 (NHE1 and NHE2) are present in BBB endothelial cells. Using microspectrofluorometry and the pH-sensitive dye BCECF, we found that hypoxia (2% O(2), 30 min), aglycemia (30 min), and AVP (1-200 nM, 5 min) significantly increased CMEC Na/H exchange activity, assessed as Na-dependent, HOE-642-sensitive H(+) flux. We found that AVP stimulation of CMEC Na/H exchange activity is dependent on intracellular Ca concentration and is blocked by V(1), but not V(2), vasopressin receptor antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation.

Exacerbated brain edema in a rat streptozotocin model of hyperglycemic ischemic stroke: Evidence for involvement of blood-brain barrier Na-K-Cl cotransport and Na/H exchange.

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood-brain barrier (BBB) Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

Histological and cognitive alterations in adult diabetic rats following an episode of juvenile diabetic ketoacidosis: Evidence of permanent cerebral injury.

Evidence suggests that diabetic ketoacidosis (DKA) may cause subtle cognitive alterations in children but the mechanisms are poorly understood. Acute DKA is associated with reactive astrogliosis and microglial activation in a rat model. Whether these inflammatory changes permanently alter brain histology is unknown. We aimed to determine whether DKA results in permanent alterations in brain histology and whether these changes are associated with cognitive deficits in a rat model. We induced diabetes in juvenile rats with streptozotocin at 4 weeks of age. We induced DKA in one group (n=21) at 5 weeks of age and compared this group to rats with diabetes without DKA episodes (n=13). Beginning at 7 weeks, rats underwent a series of cognitive tests to evaluate memory. At 15 weeks, rat brains were harvested and examined using immunohistochemistry (IHC). In tests of novel object recognition and social recognition, both groups performed similarly, however, the DKA group performed more poorly in object-place recognition tests, suggesting alterations in hippocampal function. IHC studies demonstrated increased glial fibrillary acidic protein staining intensity in the hippocampus of DKA rats suggesting astrogliosis, and decreased NeuN positive cell counts in the cortex suggesting neuron loss. These studies demonstrate that DKA results in permanent alterations in brain microstructure in a rat diabetes model. These structural changes are associated with deficits in hippocampal function.

Estradiol reduces activity of the blood-brain barrier Na-K-Cl cotransporter and decreases edema formation in permanent middle cerebral artery occlusion.

Estrogen has been shown to protect against stroke-induced brain damage, yet the mechanism is unknown. During the early hours of stroke, cerebral edema forms as increased transport of Na and Cl from blood into brain occurs across an intact blood-brain barrier (BBB). We showed previously that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia and arginine vasopressin (AVP), factors present during cerebral ischemia, and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema in rats subjected to permanent middle cerebral artery occlusion (MCAO). The present study was conducted to determine whether estrogen protects in stroke at least in part by reducing activity of the BBB cotransporter, thereby decreasing edema formation. Ovariectomized rats were subjected to 210 mins of permanent MCAO after 7-day or 30-min pretreatment with 17beta-estradiol and then brain swelling and 2,3,5-triphenyltetrazolium chloride staining were assessed as measures of brain edema and lesion volume, respectively. Diffusion-weighed imaging was used to monitor permanent MCAO-induced decreases in apparent diffusion coefficient (ADC) values, an index of changes in brain water distribution and mobility. Na-K-Cl cotransporter activity of cerebral microvascular endothelial cells (CMECs) was assessed as bumetanide-sensitive K influx and cotransporter abundance by Western blot analysis after estradiol treatment. Estradiol significantly decreased brain swelling and lesion volume and attenuated the decrease in ADC values during permanent MCAO. Estradiol also abolished CMEC cotransporter stimulation by chemical hypoxia or AVP and decreased cotransporter abundance. These findings support the hypothesis that estrogen attenuates stimulation of BBB Na-K-Cl cotransporter activity, reducing edema formation during stroke.

Bumetanide reduces cerebral edema formation in rats with diabetic ketoacidosis.

The mechanisms responsible for cerebral edema formation in diabetic ketoacidosis (DKA) are not well understood, although evidence suggests ischemia as a contributing factor. Previous studies have shown that the Na-K-Cl cotransporter of cerebral microvascular endothelial cells and astrocytes is a major participant in ischemia-induced cerebral edema in stroke. The present study was conducted to test the hypothesis that the Na-K-Cl cotransporter also contributes to cerebral edema in DKA. Sprague-Dawley rats were administered streptozotocin to induce DKA, and then cerebral edema was assessed by determination of apparent diffusion coefficients (ADC) with magnetic resonance diffusion-weighted imaging. Cerebral ADC values in DKA rats were significantly reduced in both cortex and striatum compared with non-DKA control rats, indicating the presence of cerebral edema. Intravenous administration of bumetanide to DKA rats abolished the drop in cortical ADC values, while having no significant effect in the striatum. Insulin and saline treatment had no effect when given after bumetanide but increased both cortical and striatal ADC values when given before bumetanide. Evidence is also presented here that acetoacetate and beta-hydroxybutyrate stimulate brain microvascular Na-K-Cl cotransporter activity. These findings suggest that the Na-K-Cl cotransporter contributes to brain edema in DKA.

Levels of S100B in brain and blood of rats with diabetic ketoacidosis.

Diabetic ketoacidosis (DKA) frequently causes subtle brain injuries in children. Rarely, these injuries can be severe and life threatening. The physiological processes leading to brain injury during DKA are poorly understood. S100B is a calcium-binding protein secreted by astrocytes. Elevated serum S100B levels are documented in several types of brain injuries. S100B may have either neuroprotective or neurotoxic effects, depending upon the concentration. We undertook the current studies to measure alterations in S100B production and secretion during DKA. We measured serum S100B concentrations in juvenile rats during and after DKA, and used immunohistochemistry to measure S100B expression in the hippocampus, cortex and striatum. Compared to levels in both normal and hyperglycemic control rats, serum S100B levels during DKA were significantly reduced. Serum S100B gradually rose after DKA, returning to levels of hyperglycemic controls by 72 h. S100B expression in the hippocampus was also significantly reduced 24h after DKA. There were no significant changes in S100B expression in other brain regions. Our findings contrast with those for other types of brain injuries in which both serum S100B levels and astrocyte S100B expression are typically elevated. These data suggest that serum S100B measurement cannot be used as an indicator of brain injury during DKA. Whether reduced S100B production or secretion is involved in the pathogenesis of DKA-related brain injury should be investigated.

Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke.

Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.